Hepatitis delta virus (HDV) encodes only a single protein, the hepatitis delta antigen (HDAg), which is expressed as two molecular forms (large and small) with different functions in viral replication. Compared with small antigen, large antigen has a 19 residue carboxyl terminal extension. Antibodies that recognize a large antigen-specific epitope within this carboxyl extension, or an epitope shared by both large and small antigens (total antigen), were used in immunohistochemical studies of liver sections from superinfected woodchuck carriers of woodchuck hepatitis virus. There were no differences in the subcellular distributions of large and total antigens, with both generally present only in nuclei of hepatocytes. Rare cells demonstrated cytoplasmic staining. Complete or partial granular nucleoplasmic staining with stained nucleoli was the most common pattern observed. Within 31 days of infection, 0.1% to 19% (mean = 7.4%) of all hepatocytes contained antigen. The proportion of these nuclei containing large antigen ranged from 0 to 100% (mean, 39%), and increased during the first month of infection. The number of antigen-positive nuclei and the proportion staining for large antigen were reduced with progression to chronicity, correlating with reductions in the level of viremia. Thus, the large hepatitis delta antigen shares a common subcellular distribution with small antigen and is found in an increasing proportion of the nuclei of infected cells during the course of acute infection. (HEPATOLOGY 1995; 22:1090–1100.).